I. Field of the Invention
The present invention relates to a monoclonal antibody which specifically binds to apo-B-48, hybridoma producing the same and method for measuring apo-B-48 using the monoclonal antibody. The monoclonal antibody is useful for diagnosis and therapy of hyperlipidemia and arterial sclerosis.
II. Description of the Related Art
Arterial sclerosis is one of the typical adult diseases and establishment of effective diagnosis and therapy of this disease is longed for. The causes of arterial sclerosis include increase in the amounts of various lipoproteins contained in the blood circulating the body, which accelerate deposition of cholesterol on the inner walls of blood vessels, and decrease in various lipoproteins which prevent such deposition of cholesterol.
Representative lipoproteins which are known to participate in hyperlipidemia include chylomicron (CM), chylomicron remnant (CM remnant) which is an intermediate metabolite of CM in the blood, very low density lipoprotein (VLDL), very low density lipoprotein remnant (VLDL remnant) which is an intermediate metabolite of VLDL in the blood, low density lipoprotein (LDL) and high density lipoprotein (HDL).
CM remnant, VLDL remnant and the like are called remnant-like particles (RLP). The remnant-like particles as well as LDL are lipoproteins which transport cholesterol to the blood vessel walls. Therefore, to decrease the blood levels of these lipoproteins is a direct therapy of arterial sclerosis. On the other hand, since HDL, for example, has a function to withdraw cholesterol from the lesion of arterial sclerosis, to increase the blood level of HDL is useful for the therapy of arterial sclerosis.
Among the above-mentioned lipoproteins, RLP has been reported to participate in expression of arterial sclerosis due to postprandial hyperlipidemia, and is now thought as an important risk factor of arterial sclerosis. Known apolipoproteins constituting RLP include apo-C, apo-E, apo-A-I, apo-B-100 and apo-B-48.
One of the known methods for measuring blood level of RLP is the RLP-C (remnant-like particles cholesterol) method. In the RLP-C method, anti-apo-A-I monoclonal antibody and anti-apo-B-100 monoclonal antibody are immobilized on a gel, and the gel is reacted with a test sample, followed by removal of the lipoproteins bound to these antibodies by centrifugation. Thereafter, the amount of the lipoprotein remaining in the supernatant is measured in terms of the amount of cholesterol.
Apo-A-I occurs as a major apolipoprotein of CM and HDL, and apo-B-100 occurs as a major apolipoprotein of VLDL, VLDL remnant and LDL.
Thus, theoretically, CM and HDL are bound to the anti-apo-A-I monoclonal antibody, and VLDL, VLDL remnant and LDL are bound to anti-apo-B-100 monoclonal antibody. Thus, in the RLP-C method, these lipoproteins are removed.
The anti-apo-B-100 monoclonal antibody specifically recognizes the region of the 2291st to 2318th amino acids, so that it does not recognize apo-B-48 which consists of the 1st to 2152nd amino acids of apo-B-100. Therefore, the lipoprotein which contains apo-B-48 as an apolipoprotein but does not contain apo-B-100 as an apolipoprotein is measured by the RLP-C method.
The structure of the epitopes on the apolipoproteins and the homigenity of the epitopes in the plasma lipoprotein population still remain unclear.
In the RLP-C method, the above-mentioned removal of the various lipoproteins by binding the lipoproteins to the gel is indispensable. However, this step is troublesome and has poor reproducibility because the degree of removal varies from run to run. Therefore, the measured values are not very reliable.
Apo-B-48 is contained as an apolipoprotein in CM and CM remnant. CM is quickly converted to CM remnant after eating by lipoprotein lipase and the CM remnant is taken into the liver through remnant receptors existing in the liver, so that CM does not exist in the blood when fasted. That is, Chylomicrons are lipoproteins synthesized exclusively by the intestine to transport dietary fat and fat-soluble vitamins. Among the apoliporoteins found in chylomicrons, such as apo-B-48, apo-A-I, apo-A-IV and Cs, only the synthesis of apo-B-48 is required for the assembly of chylomicrons. Apo-B-48 is a 2152 amino acid long polypeptide translated and pooled in the adult intestine from the same gene as apo-B-100 by the action of a series of enzymes. During circulation in the blood, chylomicrons are exposed to lipolysis and apolipoprotein exchange, and are converted into “chylomicron remnants”. Therefore, if there is a monoclonal antibody which specifically recognizes apo-B-48, CM remnant alone can be directly measured by using the blood when fasted as a test sample as a risk marker for postprandial lipidemia and/or arterial sclerosis. This makes it possible to accurately diagnose hyperlipidemia and, in turn, is useful for diagnosis and therapy of arterial sclerosis.
Apo-B-48 has the same amino acid sequence as a part of the amino acid sequence of apo-B-100. The amino acid sequences of both apo-B-100 and apo-B-48 are known (Nature, Vol. 323, p.738, October, 1986). An anti-apo-B-48 antiserum is also known (Journal of Biological Chemistry, Vol. 265, No. 15, p.8358, 1990; Journal of Biological Chemistry, Vol. 267, No. 2, p.1176, 1992; and Clinical Science, Vol. 85, p.521, 1993).
However, a monoclonal antibody which specifically binds to apo-B-48 alone has not been reported. Moreover, a reference (RINSHO KENSA, Vol. 40, No. 9 (1996), p. 1025, left column, line 8 from the bottom) describes that it is theoretically difficult to produce an antibody which specifically reacts with apo-B-48 alone.